In recent years, in the fields of production of medicines, gene therapy, regenerative medicine, immunotherapy or the like, it is required to culture efficiently a large amount of cells, tissues, microorganisms or the like in an artificial environment.
Under such circumstances, a large amount of cells is cultured automatically in a closed system by using a gas-permeable culture bag.
When cells are cultured for a long period of time, it is required to take photographs of cells in a culture bag at prescribed time intervals, thereby to observe periodically the number or the state of the cells. At this time, it is of crucial importance to obtain stable photographed images without fail.
However, since some culture liquids in a culture bag foam like soapy water, bubbles are also photographed when cells are photographed, and as a result, adequate counting or observation becomes impossible.
There are two main reasons that bubbles are generated in a culture bag. One reason is that a culture liquid in which oxygen and carbon dioxide have been dissolved in advance is enclosed in a culture bag. Therefore, when a culture liquid that has been stored in a refrigerator before use is warmed for use, these gases that have been dissolved may be generated in the form of bubbles. Another reason is that a film that forms a culture bag can pass oxygen and carbon dioxide therethrough. Accordingly, when a culture liquid in a culture bag is stirred by shaking a culture bag or by other methods in order to homogenize the culture liquid or for other purposes, the culture liquid foams by gases that have entered from outside through the film, and then bubbles are generated.
Therefore, as long as the thus generated bubbles are removed from an observation range, the bubbles are photographed when the inside of the container is photographed. As a result, a stable image cannot be obtained, leading to erroneous observation.
The constitution of a conventional culture bag observation apparatus and the manner of cell observation using such a conventional culture bag are shown in FIG. 12 and FIG. 13.
As shown in FIG. 12, in a conventional culture bag observation apparatus, a culture container 3′ in which a culture liquid 2′ containing bubbles 1′ is enclosed is mounted on a mounting table 4′. The observation area of the culture container 3′ is irradiated with light emitted from a lighting 9′, and a photograph of cells in the container is taken by a photographing means 8′.
As mentioned above, since oxygen or carbon dioxide is dissolved in a culture liquid in advance, as shown in FIG. 13(a), these gases may be generated as bubbles. Such bubbles may often be relatively small. However, when an enlarged photograph of cells is taken by means of the photographing means 8′ that is provided with a microscope and a camera, even small bubbles are photographed as big bubbles, and as a result, it greatly affects the observation. Therefore, even if small bubbles are generated, observation of cells could not be conducted adequately.
In the cell culture, a culture liquid in the culture bag is stirred. If such stirring is conducted, as shown in FIG. 13(b), gases that have passed through the film forming the culture bag foam by stirring, and as a result, bubbles are generated in the container. Such bubbles are relatively large, and often they are agglomerated. If cells are automatically photographed by the photographing means 8′, it was almost impossible to conduct observation of cells.
Here, as the method for removing bubbles from a liquid, as stated in Patent Document 1, for example, a method can be given in which a pressure difference is provided in the flow path of the liquid in the container, and move of the bubbles is controlled, thereby to remove the bubbles. Further, as stated in Patent Documents 2 and 3, a method can be given in which bubbles in the liquid is removed by imparting ultrasonic vibration. Further, Patent Document 4 discloses that, by controlling a flow-control actuator, an additional culture liquid is supplied to a cell culture chamber, whereby bubbles are removed.
Patent Document 1: WO2007/077607
Patent Document 2: JP-A-2004-198356
Patent Document 3: JP-A-2009-31173
Patent Document 4: JP-A-H11-507229
However, the method described in Patent Document 1 is a method in which bubbles are removed while flowing a liquid. Therefore, it is not suited to cell culture utilizing a culture bag. By this method, it is impossible to observe the cells inside the culture bag adequately. Further, when ultrasonic wave is used as in the method disclosed in Patent Documents 2 and 3, cells may be adversely affected. If bubbles are removed by using a culture liquid to be added as disclosed in Patent Document 4, cells are also removed from the observation area simultaneously. Therefore, this method cannot be applied for the observation of cells.
As the method for removing bubbles from a liquid, in addition to the methods mentioned above, a method is conceived in which the temperature is lowered, the pressure is increased or the humidity is increased, relative to the state where bubbles are generated. Further, use of an anti-foaming agent is also conceivable.
However, in the cell culture using a culture bag, decreasing the temperature when removing bubbles in the culture bag causes the cell proliferation efficiency to be lowered. The pressure can be increased when a relatively small culture bag is used when cell culture starts, etc. However, when a large culture bag is used, increasing the pressure leads to an increase in size of an apparatus. Further, in the case of increasing the humidity, small bubbles are not generated easily, and hence, the amount of gases transmitted from the outside of the culture bag can also be reduced. Accordingly, the amount of large bubbles generated by stirring a culture liquid can also be reduced. However, this method causes molds to be generated easily. An anti-foaming agent may adversely affect the survival or proliferation of cells, and hence is not suited to be added to a culture liquid.